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This powerful technology has been used to pre5606
Downloaded from http://jvi.asm. arenavirus G1 and G2 proteins are cleaved by SKI-1/S1P (6.org/ on November 12. and Chapare.1 Marina L. urine. Guanarito. The utility of the system is demonstrated by generating Junin viruses which encode a glycoprotein precursor (GPC) containing the following: (i) the wild-type (SKI-1/S1P peptidase) cleavage site. 11
Efﬁcient Reverse Genetics Generation of Infectious Junin Viruses Differing in Glycoprotein Processing †
´ Cesar G. 1600 Clifton Road. As is commonly the case with many other negative-stranded RNA viruses.gov. 33). SKI-1/S1P is localized mainly in the Golgi cisternae.JOURNAL OF VIROLOGY. Atlanta.1128/JVI. are associated with rapidly progressing severe hemorrhagic fever with a high rate of case fatality in various regions of South America. Machupo. and Chapare. and feces of infected rodents (34.1 Eric Bergeron. The pathogenic New World group of arenaviruses includes Junin. and lymphocytic choriomeningitis virus (LCMV). it does have associated toxicity. rapid onset of severe disease.200 nucleotides [nt]) encodes the matrix protein (Z) and the viral polymerase (L). although a live attenuated Junin vaccine (Candid 1) has been in use in Argentina for some years. which is associated with severe neurologic disease in immunocompromised individuals and fetal abnormalities. Khristova. 2011 by guest
* Corresponding author. while ribavirin has been shown to be of some value for treatment when used early in the course of arenavirus infections. In addition. reverse genetics systems have been successfully developed for several negative-stranded RNA viruses. It is unclear why these evolutionarily distinct HF-associated viruses use SKI-1/S1P rather than the more common furin-like PCs. Centers for Disease Control and Prevention. Arenaviruses exist in rodent reservoirs. The L segment ( 7. E-mail: stn1@cdc.400 nt) encodes the glycoprotein precursor (GPC) and the nucleoprotein (N). No FDA-approved vaccine exists for any of the HF-associated arenaviruses. In contrast to the wild-type virus. Of particular note among the Old World group of arenaviruses is Lassa virus. Sabia. or (iii) a cleavage site where the SKI-1/S1P motif (RSLK) is replaced by a furin cleavage site (RRKR). Junin. † Supplemental material for this article may be found at http://jvi . but one can hypothesize that these viral glycoproteins may have structural or functional constraints that prevent utilization of furin-like PCs for this important glycoprotein maturation step. Fax: (404) 639-1118. p. This robust. and the main natural host of JUNV is Calomys musculinus. Nichol1*
Special Pathogens Branch1 and Biotechnology Core Facility Branch.asm. The family Arenaviridae contains several viruses associated with severe human disease (9).00 0 doi:10. Rollin. MS G-14. which is associated with hemorrhagic fever (HF). while the S segment ( 3. family Bunyaviridae (40). arenavirus GPC proteins are cleaved at speciﬁc amino acid signatures by a proprotein convertase (PC) to generate the mature G1 and G2 surface glycoproteins (9). The threat of natural or deliberate outbreaks associated with these viruses makes the development of preventive or therapeutic measures important. Sabia. 41). Centers for Disease Control and Prevention. and limited vaccine and antiviral options have resulted in these HF viruses being placed on the select agent list of potential bioterrorism threat agents. Here we describe a Junin virus functional minigenome system and a reverse genetics system for production of infectious Junin virus. The arenaviruses are enveloped viruses with negative-strand bisegmented genomes which encode four viral proteins in an ambisense orientation.2 ´ ˜ Pierre E. Phone: (404) 639-1115. The associated aerosol infectivity. which is where the virus GPC is most likely cleaved (22). June 2009. 22. Junin virus (JUNV) has been known to be associated with the Argentine HF outbreaks since the 1950s (31). Atlanta. Virus transmission to humans is usually linked to contact with the blood. and PC7) for this cleavage event (38).
. Junin virus lacking a GPC cleavage site replicated within successfully transfected cells but failed to yield infectious virus particles. Published ahead of print on 25 March 2009. Over the last two decades. In contrast. although the virus has also been found in other diverse rodent species (15. PACE4. This conﬁrms observations with other arenaviruses suggesting that GPC cleavage is essential for arenavirus infectivity. Machupo. The two-plasmid. genus Nairovirus. 5606–5614 0022-538X/09/$08. Mailing address: Special Pathogens Branch. Unlike most negative-stranded RNA viruses which utilize basic amino acidspeciﬁc PCs commonly referred to as furin-like proteases (PC5. highly efﬁcient system involves transfection of cells with only two plasmids which transcribe the virus S and L antigenomic RNAs. infectious Junin virus which encoded GPC cleaved by furin-like proteases was easily generated. Albarino. GA 30333. Georgia 303332
Received 7 February 2009/Accepted 11 March 2009
The New World arenaviruses. furin. No.1 and Stuart T.org/. MS G-14. 1600 Clifton Road. The need for better tools to combat these diseases and more knowledge of arenavirus-associated pathogenesis remains critical. high efﬁciency aspects of this Junin virus reverse genetics system show great promise for addressing important questions regarding arenavirus hemorrhagic fever disease and for development of precisely attenuated live arenavirus vaccines. 25. (ii) no cleavage site. 28. each associated with severe HFs in various parts of South America. 83. 35).1 Bobbie Rae Erickson. Guanarito. The only other negative-stranded RNA virus known to share this unusual glycoprotein cleavage feature is Crimean-Congo HF (CCHF) virus. 29.00276-09
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Such a system would be highly valuable to address fundamental questions regarding the biology and pathogenesis of New World arenaviruses and allow development of prototypic live attenuated vaccines with precisely engineered disruptions of pathogenic properties. Reverse genetics systems for arenaviruses have been developed utilizing virus minigenomes for the study of LCMV. RNA was extracted using either RNAid (Qbiogene) or RNeasy (Qiagen) following the manufacturer’s recommendations. a generous gift from L.SDD). following the manufacturer’s protocol. We report here the successful development of a highly efﬁcient two-plasmid reverse genetics system for the generation of JUNV variants. the ampliﬁcation of full-length L could not be easily achieved. and ligated into a modiﬁed version of the V0. The terminal sequences of JUNV strain XJ13 were determined by 3 rapid ampliﬁcation of cDNA ends (RACE) and RNA ligation as described previously (1). depending on the experiment. the S RNA was ampliﬁed by RT-PCR. In these systems. To detect JUNV genomes and antigenomes. S. Cloning of full-length JUNV S RNA (3. we used primers corresponding to the following respective positions: S-fwd (nt 3207
to 3226) and S-rev (nt 250 to 270) for S RNA and L-fwd (nt 6636 to 6656) and L-rev (nt 352 to 368) for L RNA. or 24well plates. Ligated RNAs were further puriﬁed and used as templates in RTPCR analyses. 23. BSR-T7/5 cells constitutively expressing T7 polymerase were a generous gift of K. 37). 8. puriﬁed using the RNeasy kit (Qiagen). transcription of the virus minigenome version of the S RNA segment is produced either in the cytoplasm by the T7 RNA polymerase or by the polymerase I in the nucleus. For RNA ligation. NC_005080. Although we obtained several clones of full-length S. Eventually. reverse genetics have been developed to allow the rescue of infectious recombinant LCMV. To check replication of GFP minigenomes. Lassa virus. SRD12B cells (SKI-1/S1P deﬁcient) were propagated as described previously (4). mounted with 4 . but the yield was small. Cloning of the JUNV S and L followed the standard T7-based strategy we had successfully applied to Rift Valley fever RNA segments previously (1. Indeed. Alternatively. Infected cells and/or viral supernatants were harvested at 3 to 5 days postinfection (p. digested with BsmBI. and chicken anti-rabbit conjugated with Alexa Fluor 488. tubes containing the resulting noninfectious lysates were surface decontaminated and then transferred into a BSL-2 laboratory.114 nt). virus propagation. i. following the manufacturer’s protocol. These experiments demonstrated that JUNV GPC cleavage is essential for the release of infectious virus particles but that furin-like PCs can effectively be substituted for SKI-1/S1P in the cleavage process and generation of infectious virus. we checked expression of JUNV proteins in transfected cells by immunoﬂuorescence assay (IFA) using a rabbit serum raised against JUNV or monoclonal antibodies (MAb) against N that were described previously (36).VOL. treated with Triton 1%. and biosafety. were designed to transcribe the full-length antigenomic copies of JUNV S and L RNAs and contained two nonviral nucleotides. possibly due to the strong RNA secondary structure that somehow was an impediment to the cDNA synthesis and/or the ampliﬁcation. due to the total length of JUNV L RNA (7. we managed to amplify a full-length L copy. Invitrogen). and the cloning process proved unsuccessful. and corrected several nucleotide changes in consecutive steps while keeping some minor mutations as genetic markers. For 3 RACE. we randomly chose one of them. 83. 546. More recently.GPC:GFP. 16). and then several nucleotide changes corrected in consecutive steps while keeping some minor mutations as genetic markers.asm.). pJunS. Variants of the full-length L and S clones were constructed by classic PCR mutagenesis which would encode altered virus protein motif changes. The plasmids described above were transfected in monolayers of BSR-T7/5 growing in 6-well. Based on this ﬁnding. 13. The ﬁnal plasmid constructions.6diamidino-2-phenylindole (DAPI). Basically. Transfection of plasmid DNA and viral rescue. required for the efﬁcient function of the T7 promoter and the hepatitis delta virus ribozyme. 12. The robust nature of the system likely reﬂects the simplicity of the two-plasmid procedure. pJunS and pJunL. and then ligated with T4 RNA ligase (NEB) using standard conditions. The resulting clone. none of them presented a sequence identical to the viral RNA. and 594 (Molecular Probes. viral RNAs were extracted from supernatants or from Vero E6-infected cells as described above. and photographed with an inverted microscope. After these initial attempts. All primers were designed according the sequences available in GenBank for JUNV XJ13 (L. 2011 by guest
. and sequenced using standard protocols (ABI). An additional problem emerged in the cloning process due to the unstable nature of the resulting clones containing JUNV L sequences. A. The resulting RT-PCR products were separated by electrophoresis in agarose gels. one of the L clones that was well tolerated in the bacteria was chosen and sequenced to completion.. and L and N expression initiates transcription and replication from the primary transcripts (18.org/ on November 12. Moreover. Additional technical details may be supplied upon request.i. Transfection medium was replaced 6 to 14 h later with regular DMEM containing 5% FBS. the GPC cleavage site (RSLK) in pJunS was changed to AAAA (pJunS. we ampliﬁed JUNV L RNA in two halves. the cloning process was more complicated and took several additional steps. 2009
REVERSE GENETICS GENERATION OF INFECTIOUS JUNIN VIRUSES
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cisely address numerous questions regarding viral pathogenesis and replication and to develop prototypic live attenuated vaccines (5. Conzelmann (Max-vonPettenkofer-Institut.
MATERIALS AND METHODS Cell culture. carries the N gene in positive sense and GFP in negative sense. most of the clones that were well tolerated in the competent bacteria contained severe mutations such as stop codons and frameshift mutations in the L open reading frame (ORF). 12-well. Plasmid construction. primary transcription of the LCMV genome was driven by either the polymerase I or T7 polymerase in the presence of support plasmids expressing L RNA polymerase and N (11.411 nt) was a relatively straightforward process. Invitrogen) once every other passage. Virus propagation was routinely done by infecting conﬂuent monolayers of Vero E6 cells in 25-cm2 ﬂasks. RNAs were polyadenylated using the poly(A) polymerase tailing kit (Epicentre) by following standard protocols and then puriﬁed using the RNeasy kit (Qiagen). and Tacaribe virus transcription and replication mechanisms. we initiated a project to attempt to design an efﬁcient reverse genetics system for the rescue of infectious JUNV variants. From the beginning. viral RNAs were treated with tobacco acid pyrophosphatase (Epicenter). Manipulations of wild-type (wt) JUNV or recombinant JUNV (rJUNV) were performed within a biosafety level 4 (BSL-4) containment laboratory. 5 G and 3 C. 32). we replaced the GPC ORF in pJunS by the enhanced green ﬂuorescent protein (GFP) ORF by conventional cloning techniques.e. washed with PBS. As expected. For this reason.0 transcription plasmid. an Old World arenavirus. Ten microliters of in vitro polyadenylated RNA was used as a template in reverse transcription-PCR (RT-PCR) analyses by using the SuperScript III one-step RT-PCR system with Platinum Taq High Fidelity (Invitrogen). Additional technical details may be supplied upon request. With these systems. BSR-T7/5 cells were propagated in the presence of 1 mg/ml of the selective agent G418 (Geneticin. BSR-T7/5 and Vero E6 cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and penicillin-streptomycin at the standard concentration suggested by the manufacturers. NC_005081). digested with appropriated restriction enzymes and ligated into the transcription plasmid. goat anti-mouse.SKI and RRKR [pJunS-Furin]). we took an approach similar to that used to obtain functional S clones. Parallel RT-PCR analyses were done using an oligo(dT)-containing primer in combination with primers speciﬁc for S and L RNAs. 20. sequenced it to completion. Germany). Ball at the University of Alabama at Birmingham. The utility of the system was validated by generation of JUNV variants with altered GPC cleavage properties. The following commercially available secondary antibodies were routinely used: goat anti-rabbit. using LT1 transfection reagent (Mirus) in a ratio of 3:1 or 5:1 reagent to DNA. 21. Virus supernatants were harvested for RNA extraction using TriPure (Roche) at a ratio of 5:1. 7). IFAs were done with standard protocols and photographed with an Axio-Imager
Downloaded from http://jvi. the supernatant was removed and cells were ﬁxed with 3% formaldehyde in phosphate-buffered saline (PBS). further puriﬁed by using the QIAquick gel extraction kit (Qiagen). Determination of terminal sequences. Munich. The SDD polymerase motif in pJunL was changed to AAA (pJunL. Given the substantial genetic and biologic differences between the LCMV and the New World arenaviruses associated with HF disease. Brieﬂy.

and stored at 70°C. S. with AAA (Fig. Interestingly.i. Resolved proteins were transferred onto nitrocellulose membranes by using dry electrotransfer with an iBlot apparatus (Invitrogen) before being incubated with JUNV G2 MAb (QD04-AF03) (36). In order to generate an appropriate negative control plasmid for the reverse genetics experiments. we cloned full-length antigenomic copies of JUNV S and L RNAs in a T7-based system described previously (1. 1.GPC:GFP) was modiﬁed to inactivate polymerase activity by replacing the core active motif. the predicted panhandle structures for the terminal 19 nt reveals perfect complementarity for the L RNA segment termini but not for the S RNA segment (Fig. while the inactive pJUNL. 2A). Six nucleotide differences were found (Fig. In order to generate recombinant viruses. while no additional 5 G was found at the end of the L RNA molecules. NC_005081). and infecting fresh monolayers of Vero E6 cells in 75-cm2 ﬂasks. As previously observed (13. Proteins were separated by electrophoresis on a NuPAGE 4 to 12% gradient Bis-Tris gel in morpholineethanesulfonic acid (MES) buffer (Invitrogen) under reducing conditions. the JUNV strain XJ13 RNA terminus sequences were directly determined and compared to those primer bind sites reported in the JUNV entries in GenBank (L. (t 0). The S RNA sequence differed in two positions (7AxC and 9TxG) that correspond to the 5 end. transfection of the pJunS. The defects in the nonfunctional L clones frequently included single-base deletions which altered the reading frame or mutations creating stop codons within the ORF. NC_005080. one that supported GFP production in cells transfected with pJunS. Infected cells were analyzed 3 to 5 days later by IFA as described above. 7). Before starting the cloning process. one of the active L clones (i. The functional L plasmid clone selected for subsequent use encoded an L polymerase which differed from the authentic virus L polymerase by only three amino acid positions (see Table S1 in the supplemental material). Vero E6 cells growing in 25-cm2 ﬂasks were infected with the wt JUNV.5 ml of the 3 ml of supernatant was removed from the well of the six-well plate of transfected BSR-T7/5 cells 3 to 5 days posttransfection. As shown in Fig.
Downloaded from http://jvi. NC_005080. generating plasmid pJUNL.
(Zeiss) direct microscope or a Leica DM6000B confocal microscope. and g signiﬁes an extra G found at the 5 end of approximately 25 to 50% of the population of JUNV S RNAs analyzed. The JUNV entries in GenBank (L. VIROL.
. These samples were diluted in DMEM and titrated. we made two additional constructions in order to test their functionality. Transfection of these plasmids into T7-expressing BSR-T7/5 cells was expected to generate primary transcripts that correspond to JUNV complete S and L anti-genome RNAs that may be translated to produce sufﬁcient N and L proteins to initiate viral RNA replication. actin MAb (GenScript). diluting it into 5 ml of medium. i.e.2 to 0.SDD.5608
ALBARINO ET AL. Using 3 RACE and RNA ligation approaches. 5.asm. and 7 days p. 2A). This construction allowed us to test the functionality of about 60 L clones and choose the active clones before sequencing them to completion. while the L RNA sequences differed in four positions that correspond to both the 5 end (7AxC and 9TxG) and the 3 end (7107AxT and 7109TxG). 1B). Western blot analysis. pJunL and pJunS. The ﬁrst sample was taken at 1 h p. and plaques were detected by immunochemistry and by staining with crystal violet using previously described protocols (39). The ﬁnal constructions. NC_005081) include segment terminus sequences. SDD. but these are identiﬁed as primer binding sites. followed by the T7 terminator. 2) and was used as a control in all subsequent experiments. followed by the addition of 5 ml of tissue culture ﬂuid.GPC:GFP mini-replicon along with the active pJUNL clone resulted in GFP-expressing cells. For viral rescue. Cell lysates were -irradiated with 2 106 rad before aliquots of 50 l were denatured for 15 min at 65°C with denaturation buffer. First. Bands were revealed with SuperSignal West Dura chemiluminescence substrate (Pierce) and detected with the FluorChem HD2 imaging system (Alpha Innotech). (B) Predicted panhandle structures for both S and L genomic RNAs. Nucleotide differences are shown underlined. After 1 h of adsorption. JUNV RNA genome termini. the monolayers were washed three times with PBS and supplied with DMEM containing 2% FBS.. The 3 RNA termini of arenavirus genomic and antigenomic RNAs contain critical regulatory regions important for polymerase binding and encapsidation processes involved in virus transcription and replication.
~
J. it was important to experimentally determine these terminal sequences for JUNV S and L RNA segments as we could ﬁnd no evidence in GenBank entries or in the literature that this had been done previously. have the viral insert ﬂanked by the T7 promoter and the hepatitis delta virus ribozyme. 2011 by guest
RESULTS Determination of terminal sequences of JUNV genome RNAs. the sequences were not determined but merely represent the sequences of the primers used to amplify the PCR products. 0. about 25 to 50% of the S RNA molecules had an additional 5 G relative to the complementary 3 end.e.01.i. 14).SDD did not show any sign of activity (Fig. (A) Virus RNA terminal sequences were experimentally determined by 3 RACE and RNA ligation (Exp) and are shown in comparison with available sequences from GenBank (GB).2 (Alpha Innotech). All transfection reactions with the potential to generate infectious virus were carried out in the BSL-4 laboratory.
FIG. S. Following construction of the initial virus L clones. rJUNV.0. followed by digestion with PNGase F (500 U) or recombinant EndoH (1. or RRKR mutant viruses (rJUNV-Furin) at a multiplicity of infection of 0. or successive passages were done by transferring 1 ml of the 5 ml of supernatant from the 25-cm2 ﬂask.. Infected cells were lysed in mammalian protein extraction reagent (Pierce) with Halt protease inhibitor cocktail (Pierce). 3. Four out of the 60 L clones demonstrated polymerase activity. Densitometry of the bands was analyzed with AlphaEaseFC software version 6.
Replication of JUNV minigenomes and rescue of recombinant virus. The orientation of the resulting plasmid is such that GFP would be produced only when RNA replication occurred (Fig. Virus growth curve. 1A).000 U) for 1 h at 37°C (New England Biolabs). 2B. This was diluted to 1 ml of medium and used to infect a Vero E6 cell monolayer in a 25-cm2 ﬂask (ﬁrst passage). a ﬂuorescent reporter gene was inserted into the S template by replacing the GPC ORF by enhanced GFP (pJunSGPC:GFP).org/ on November 12. and subsequent samples were collected at 1. and anti-mouse horseradish peroxidase-coupled secondary antibody (Pierce).

3C). For this purpose. 83.VOL. These were not observed in the cell monolayers transfected with the negative control. 2. and the virus genome was sequenced to verify that the recombinant virus S and L segment sequences were identical to those of the template plasmids. recombinant virus master stocks were prepared. JUNV proteins were detected 5 dpt with anti-Junin rabbit serum and anti-rabbit Alexa Fluor 594 (red stain).
With these basic tools successfully developed. 3C). indicating successful rJUNV replication and spread. Transfection of cells with pJUNS-Furin plus pJunL did successfully yield infectious virus (Fig. 3). and PC7) utilized by other negative-stranded RNA viruses (6. (B) Functional L protein required for minigenome replication. 3C). (A) Schematic of plasmids indicating resulting primary transcripts. supernatant from the pJUNS. cells were analyzed for evidence of virus production by staining them with JUNV reactive antibody (Fig. Transfected cells were ﬁxed 3 dpt. This is exactly the result obtained. (C) Functional L protein required for virus replication and spread. as a negative control.SDD. as evidenced by immunoﬂuorescence staining for JUNV following passages in Vero E6 cells. the rJUNV-Furin virus still grew to a lower titer by the second Vero E6 cell passage (Fig. 22.SDD (left). counterstained with DAPI and photographed with UV light in the GFP range (green cells). family Bunyaviridae) share the unusual feature of having the major cleavage step which generates the virus mature surface glycoproteins mediated by the SKI-1/S1P protease rather than the furin-like proteases (PC5. was replaced by a canonical furin-like cleavage site. In addition. The number of positively staining cells was signiﬁcantly less than that observed with the wt (rJUNV) rescued in parallel experiments (Fig. 2009
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FIG. 3B). indicating the robust nature of the technique and the high level of efﬁciency of a system requiring the transfection of cells with only two plasmids.SDD (Fig. the procedure has been repeated multiple times with 100% success. then it was anticipated that transfection of cells with this plasmid (in conjunction with pJunL) would result in virus replication but not in the release of infectious virus.GPC:GFP and pJunL (right) or pJunL. Cells supporting active virus replication were observed in the monolayers of cells transfected with pJUNSSKI plus pJunL but not in cells transfected with pJUNS. 2011 by guest
. Proof of system utility by generation of virus GPC cleavage mutants. However. The rJUNV and rJUNV-Furin genome sequences were veriﬁed and shown to be identical except for the engineered cleavage site mutation. 2C). Rescue of infectious virus was demonstrated by infection of fresh monolayers of Vero E6 cells with supernatant off the positively transfected cell monolayers. 33. A version of the pJunS plasmid was designed where the virus GPC SKI-1/S1P cleavage signal (RSLK) was abolished (pJUNS. BSR-T7/5 cells were transfected with the full-length plasmids. conﬁrming
Downloaded from http://jvi. However. Following the initial successful rescue of infectious virus. attempts were made to generate infectious JUNV entirely from plasmid DNA. Cells were counterstained with DAPI and photographed with UV light in a regular upright microscope. PACE4. The majority of cells were demonstrated by IFA to be infected. data from experiments utilizing SKI-1/S1P cell lines deﬁcient in SKI-1/S1P have strongly suggested that GPC cleavage by SKI-1/S1P is strictly required for Lassa virus and CCHF virus infectivity (4. If this cleavage event was strictly required for generation of infectious virus particles. Perhaps there is some structural or functional constraint on these glycoproteins which prevents furin utilization for this cleavage event. another version of pJunS was constructed where the SKI-1/S1P cleavage signal. BSR-T7/5 cells were transfected with pJunS and pJunL (right) or pJunL. However. RSLK. These results clearly demonstrated that in the context of authentic JUNV. The arenaviruses and CCHF virus (genus Nairovirus. including the marker mutations included in the plasmid construction process (GenBank entries FJ805379 to
FJ805380). the furin-cleaved mutant virus (rJUNV-Furin) appeared to grow less efﬁciently. Approximately two to ﬁve large foci of positively stained cells were seen in each well of the six-well plate. indicating that furin-like PCs could effectively replace SKI-1/S1P in the GPC cleavage process despite the strict conservation of the SKI-1/S1P motif among the GPCs of all arenaviruses. Why these evolutionarily unrelated HF-associated viruses utilize SKI-1/S1P and not furin-like PCs is an enigma. RRKR (pJUNS-Furin).SKI plus the negative control pJunL. In addition.SKI plus pJunL-transfected cell monolayers was used to initiate two blind passages on new Vero E6 monolayers with no evidence of virus growth (Fig. BSR-T7/5 cells growing in 24-well plates were transfected with pJunS.org/ on November 12. furin. inactive L polymerase (pJUNL. 3C). pJunS and pJunL or pJUNL.asm. SKI-1/S1P cleavage appears to be strictly required for the release of infectious virus particles.SDD (left). Six days after transfection. 22).SKI) (Fig. To determine if there was a functional or structural constraint preventing arenavirus GPC cleavage by furin-like PCs. some other role for SKI-1/ S1P in the virus life cycle could not be ruled out in these studies. The newly developed JUNV reverse genetics system provided the opportunity to precisely examine these issues in the context of authentic JUNV. 38).SDD).

GPC cleavage was equally efﬁcient ( 65%) based on similar ratios of uncleaved GPC to G2 bands observed for the two viruses (Fig.to 100-fold less than those for rJUNV and JUNV. 4B. we compared the growth in cell culture of the mutant virus with that of the authentic wt JUNV and the reverse genetics-derived wt virus (rJUNV). GPC was detected with a MAb anti-Junin GPC and anti-mouse Alexa Fluor 488 (green stain).i. Since we recovered rJUNV-Furin virus with a lower efﬁciency than rJUNV. pJunS. However.SKI or pJunS-Furin with pJunL or pJunL.asm.
that the growth defect was directly related to the cleavage site alteration (see Table S1 in the supplemental material). these data indicate
. infected cell lysates were analyzed by Western blot analysis using a G2-speciﬁc MAb (Fig. and virus titers were obtained by using a conventional plaque assay (Fig. (C) Virus GPC cleavage by SKI-1/S1P or furin-like PCs is required for rescue of infectious rJUNV. 1. lane 2) resulted in a mobility shift of the 64-kDa form of GPC to an 49-kDa form.
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ALBARINO ET AL. Treatment with endoglycosidase H (EndoH) to remove high levels of mannose sugars on the wt rJUNV lysate (Fig. In contrast. 2011 by guest
FIG. lane 5 versus lane 2) revealed an increase in the amount of the 44-kDa form of G2 and a large reduction in the amount of the 35-kDa form of G2 relative to that of the 49-kDa form of GPC.SDD. In order to determine whether the defect in rJUNV-Furin resided in GPC processing. lanes 3
and 6) showed that the total amounts of GPC and G2 proteins synthesized by both rJUNV and rJUNV-Furin were approximately equal. 4B. and 7 days p. 4B.i.org/ on November 12. Comparison of the EndoH-treated lysates of rJUNV-Furin relative to those of rJUNV (Fig. BSR-T7/5 cells were transfected with pJunS. 3. Together. For instance. 4B). where maximum virus titers of approximately 105 PFU/ml were obtained about 5 to 6 days p. 4A). 3. Plasmid primary transcript is shown 3 to 5 . The efﬁciency of viral rescue was examined by focus formation in Vero E6 cells (1st and 2nd passages) and by plaque assay of virus stocks (2nd passage). Examination of the glycoproteins following removal of all Nglycans with the endoglycosidase F (EndoF) (Fig. Cells were counterstained with DAPI and photographed with UV light in a confocal microscope. 2 to 5 indicates an average of 2 to 5 large ( 50 cell) foci of infected cells/well on a six well plate. In addition. a comparison of the various glycosylated forms of the glycoproteins synthesized by rJUNV and rJUNV-Furin revealed clear differences between these viruses with regard to the modiﬁcations of their N-glycans (Fig. 4B. VIROL. and the maximum titers reached were approximately 50. The authentic JUNV and rJUNV viruses exhibited similar growth curves. lanes 3 and 6). supernatant samples were collected at 0. lanes 1 to 2 and 4 to 5). and the encoded GPC cleavage site is indicated for each plasmid. N protein was detected 3 dpt with anti-Junin rabbit serum and an anti-rabbit Alexa Fluor 594 (red stain). growth of rJUNV-Furin appeared to be slower..
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J. (A) Schematic of plasmids encoding virus GPC cleavage variants.01. Vero E6 cells were infected with a multiplicity of infection of 0. examination of nontreated lysates (lanes 1 and 4) showed that rJUNV-Furin synthesized less of the 38-kDa form of G2 than rJUNV. and the 38-kDa form of G2 shifted to an 35-kDa form. 4B. 5. (B) Replication of wt and cleavage variant JUNV in transfected cells. rJUNV-Furin virus growth is attenuated in cell culture.

Blot was reprobed with anti-actin MAb to control the amount of loaded proteins in the gel. Duplicate samples were collected and titers determined at 0. These data indicate that the strict requirement of SKI-1/S1P for generation of infectious arenavirus particles is directly related to the need for virus GPC cleavage. and 7 days p. (A) Vero E6 cells were infected with wt JUNV (triangle).i. 2011 by guest
FIG. 5. then the rJUNV-Furin should
. The absence of infectious virus released from cells successfully transfected with pJUNS. Approximately seven or eight large infected cell foci (more than ﬁve cells) were observed per ﬁeld by 3 days p. The rJUNV-Furin virus infection clearly spread in cell monolayers in the absence of active SKI-1/S1P (Fig. These elements. 5. 5. With regard to the JUNV RNA terminal sequences derived
that SKI-1/S1P cleavage can be substituted by a furin-like motif without affecting GPC production and cleavage but that this substitution leads to reduced growth of the rJUNV-Furin and differences in the N-glycan maturation of the virus glycoproteins.VOL. SKI-1/S1P-deﬁcient cells (SRD12B cells) were infected with rJUNV or rJUNV-Furin viruses.org/ on November 12. To investigate this possibility. 83. but no spread of virus or infected cell foci was observed (Fig.i. decRVKR-CMK (Fig.i. laid the necessary foundation for the infectious virus rescue attempts. 33). bottom right). As expected. 1. This success was achieved by ﬁrst determining the correct sequence of the virus RNA genome termini which contain critical promoter and encapsidation signals. EndoH resistant forms of GPC (asterisk). Glycosylated forms of GPC and G2 forms are indicated with open arrows and unglycosylated forms with black arrows. 22. JUNV proteins were detected 3 dpi using an anti-Junin rabbit serum and an anti-rabbit Alexa Fluor 546 (red stain). If the arenavirus requirement for SKI1/S1P is to cleave only GPC. The distillation of the procedure down to a simple two-plasmid system (pJUNS and pJUNL) resulted in generation of a highly efﬁcient and robust virus rescue system.asm. SKI-1/S1P-independent growth of rJUNV-Furin virus. What remains unclear is whether SKI-1/S1P is required for other aspects of the arenavirus replication cycle in addition to GPC cleavage. top left). (B) Protein lysates from Vero infected cells were examined by Western blot using anti-JUNV GPC MAbs. 3. In addition. infected cell foci were not seen in any of the negative control monolayers. followed by development of a JUNV minigenome system to allow screening of functional JUNV L polymerase-encoding clones.
FIG. The wt rJUNV could infect SKI1/S1P-deﬁcient cells. rJUNV-Furin exhibits altered virus growth and glycoprotein maturation. SRD12B cells deﬁcient in SKI-1/S1P were infected with rJUNV or rJUNVFurin in the absence or presence of 30 M the furin inhibitor decRVKR-CMK. as evidenced by virus-speciﬁc antibody staining of initial individual infected cells. rJUNV-Furin grows in SKI-1/S1P deﬁcient cells. bottom left). coupled with the construction of an appropriate negative control L polymerase (lacking the SDD core motif). 19. and G2 (triangle and circle) are also indicated. Cells were counterstained with DAPI and photographed with UV light in a regular upright microscope. indicating a strict requirement for cleavage of Lassa virus or JUNV GPC for generation of infectious virus particles (6. spread of the rJUNV-Furin virus infection in SKI-1/ S1P-deﬁcient cells was shown to be prevented by a nontoxic dose of a commonly used furin-like protease inhibitor. 2009
REVERSE GENETICS GENERATION OF INFECTIOUS JUNIN VIRUSES
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Downloaded from http://jvi. rJUNV (square) and rJUNV-Furin viruses (circle). Thirty-six hours p.
grow in SKI-1/S1P-deﬁcient cells due to cleavage of its GPC by furin-like PCs. 4. supernatants were replaced with fresh media with or without inhibitor.SKI and pJunL plasmids is consistent with earlier data (using SKI-1/S1P-deﬁcient cells). 5. 5. DISCUSSION The development of JUNV reverse genetics systems for minigenome replication and generation of infectious virus represent an important breakthrough in efforts to understand and combat infections associated with the HF-associated New World arenaviruses.

The JUNV reverse genetics system described above represents a powerful tool to precisely address questions regarding arenavirus biology and pathogenicity. family Bunyaviridae) (7). the growth of rJUNVFurin in SKI-1/S1P-deﬁcient cells (Fig. avoiding the requirement of additional protein-encoding support plasmids by relying solely on full-length plus-strand copies of the three RNA genome segments (S. but it remained unclear whether glycoprotein cleavage was solely involved or whether other steps in infectious virus production required functional SKI-1/S1P. 19. Here we demonstrated (Fig. suggesting that SKI-1/S1P GPC-cleaved virus has a ﬁtness advantage over furin GPC-cleaved virus. At ﬁrst the basis for this was unclear as biochemical analysis of the virus glycoproteins revealed that the level of GPC processing was not signiﬁcantly different between rJUNV-Furin and rJUNV. Previous ﬁndings strongly suggested that arenavirus GPC processing by the protease SKI-1/S1P was absolutely required for arenavirus growth in cell culture (6. particularly with regard to the New World HF-associated arenaviruses.SKI mutant to yield infectious virus is consistent with earlier studies which suggested that while uncleaved GPC is expressed at the cell surface. despite these cells producing active SKI-1/S1P. that the rJUNV-Furin virus was recovered less efﬁciently and grew to lower titers in Vero E6 cells than the wt rJUNV. without the need for additional expression vectors to express the nucleoprotein. Arenavirus G1/G2 heterodimers associate in three noncovalently associated subunits. an evolutionarily unrelated virus of the family Bunyaviridae (4). Based on these experiences. Why the switch in cleavage from SKI-1/S1P to furin should increase the GPC and G2 complex oligosaccharide chain content may be a matter of timing. We hypothesized that there must be some functional or structural feature of these glycoproteins which prevents cleavage by furin-like PCs which is the more common cleavage mechanism among the negative-strand RNA viruses. However. as only two plasmids are required to rescue an rJUNV. Lowen and colleagues had earlier pioneered a reverse genetic system for Bunyamwera virus (genus Orthobunyavirus. This hypothesis was proved to be incorrect by the successful generation of the infectious rJUNV-Furin where the SKI-1/ S1P cleavage site (RSLK) had been replaced by a furin cleavage site (RRKR) (Fig. which are incorporated in budding virions at the cell surface. The system has proven to be remarkably robust. M. it is possible that GPC cleavage in a different compartment as it transits the Golgi results in subtle differences in the carbohydrate chain addition during trafﬁcking from the endoplasmic reticulum to cell surface. 5) conﬁrmed that the GPC of this virus was not cleaved by SKI-1/S1P but by furinlike PCs. We now have the required tools on hand to initiate study of the impact of sequence and predicted structure variation of the RNA termini relative to these processes. This result also demonstrated that there was no additional SKI-1/S1P-dependent step (beyond GPC cleavage) essential for production of infectious virus. it is now clear that all the known arenavirus RNA termini share the feature whereby the termini of the L RNA segment have the potential to form a perfect panhandle structure. To test this. VIROL.SKI replicated successfully in transfected cells but did not yield infectious progeny virus. The observation that the rJUNV-Furin virus had reduced growth properties in tissue culture led us to examine whether switching the virus GPC cleavage from SKI-1/S1P to furin-like PCs had produced a virus with attenuated pathogenicity. In addition. as infectious rJUNV and rJUNV-Furin virus have now been consistently generated in many replicas and variations of the original protocol.org/ on November 12. and packaging efﬁciency of the S and L RNA segments. 3B). In addition. This approach also relies on sufﬁcient translation of L and N proteins from the T7-generated antigenomic transcripts to initiate virus genome replication. suggesting that GPC cleavage is also critical to generate the fusion-competent glycoprotein form. Generation of the mature virion glycoproteins by SKI-1/S1P cleavage is a feature apparently shared by arenaviruses and CCHF virus. 4B). a pilot experiment was done in which 9-dayold suckling mice (two groups of 10) were inoculated intracerebrally with either a lethal dose (400 PFU) of wt rJUNV or an
Downloaded from http://jvi.asm. 22. It was noted. 3C) in the context of actual JUNV that the protease cleavageresistant mutant pJUNS. it is not incorporated into virus particles (6. signiﬁcantly less GPC and G2 proteins with a large mannose content (EndoH sensitive) and more EndoH-resistant GPC and G2 were observed for the rJUNV-Furin. Such alterations could correlate with the growth differences observed. This novel experimental approach for arenavirus reverse genetics presents an important advantage over previously reported LCMV systems (11. We based our reverse genetic rescue of infectious virus solely on synthesis of the full-length antigenomic S and L RNAs in T7-based transcription vectors. 13. 33). 33).5612
ALBARINO ET AL. and L) to generate sufﬁcient virus proteins to initiate virus replication (24). but the termini of the S RNA segment contain mismatches that would result in an imperfect structure (30). The development of safe and efﬁcacious live attenuated vaccines for the New World HF-associated arenaviruses is an important long-term goal of our reverse genetics efforts with these viruses. The JUNV reverse genetics system allowed this point to be made in a more unequivocal manner. avoiding the use of SKI-1/S1P inhibitors or deﬁcient cell lines or pseudotype systems. These experiments clearly identiﬁed SKI-1/S1P as a critical cellular factor for production of infectious virus. 2011 by guest
. viral polymerase. whereas furin is predominantly in the trans-Golgi network. 37). family Bunyaviridae) that generated infectious virus from a system. however. The inability of the cleavage-defective pJUNS. we decided to attempt to apply the same concept to the JUNV ambisense RNA genome.
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J. These data conﬁrmed
that JUNV GPC processing is strictly required for production of infectious virus particles. or T7 RNA polymerase. replication. conﬁrming that the RRKR motif was cleaved with very similar efﬁciency (Fig. it had previously been shown that overexpression of a cleavage-resistant form of the JUNV GPC failed to induce cell-cell fusion (42). We had successfully applied the same concept for the generation of a high-efﬁciency reverse genetics system for the rescue of Rift Valley fever viruses (genus Phlebovirus. glycoprotein. The utility of the system was illustrated here by using it to examine elements of virus glycoprotein processing. 22. 19. The strict conservation of this feature suggests it has a likely role in balancing the relative transcription. These data suggest that this simpliﬁed approach should be equally applicable for the development of efﬁcient reverse genetics systems for other arenaviruses. 20. As SKI-1/S1P is located predominantly in the early Golgi.
here.